Examining the biophysics of ultrasound and contrast agent induced angiogenesis

Ischemic diseases affect more than 100 million people in the United States alone (NDFS 2011, Roger et al. 2011). Current interventions include: ablation, angioplasty, revascularization and bypass surgery. The invasive nature of these techniques excludes patients with who are not amenable to surgical intervention. For this reason, alternative methods of revascularization in ischemic cardiac muscle have been explored. Over the past two decades, cellular, molecular, and genetic therapy attempts have been made in order to find a clinically relevant treatment. Ultimately, it is the invasiveness or lack of site specificity that provides the largest obstacle for therapeutic effectiveness of angiogenic treatments. Current research suggests that ultrasound-ultrasound contrast agent treatment can be therapeutically beneficial, providing a noninvasive way to spatially and temporally target ischemic tissues. This type of angiogenic therapy can be used as an alternative to high risk percutaneous intervention or bypass graft surgery. Several studies report a reparative response to ultrasound-ultrasound contrast agent exposure and state that inertial cavitation, or microbubble collapse, is possibly required for angiogenesis to occur. Numerous small-scale studies have shown promising results; however, when large scale double blind studies were conducted, they showed limited effects. A major impediment for progress to clinical applicability is, perhaps, the lack of understanding the biophysical mechanisms that connect ultrasound-ultrasound contrast agent to neovascularization. This thesis seeks to explore the biophysics of ultrasound-ultrasound contrast agent-induced capillary angiogenesis, specifically examining the role of the ultrasound contrast agent in creating bioeffects that lead to subsequent angiogenesis. The mechanistic exploration examines both the necessity and concentration of ultrasound contrast agents, bubble dynamics, the biophysical effects of ultrasound contrast agents and several ultrasound parameters on causing/enhancing ultrasound-induced angiogenesis. A series of experiments were conducted to examine the biophysics of ultrasound and ultrasound contrast agent induced angiogenesis. The first experiment explores the effect of ultrasound contrast agents on ultrasound-induced angiogenesis with a 3 x 2 x 4 factorial study assessing survival day, infusion media (saline or ultrasound contrast agent) and acoustic pressure, respectively. Then, several exposure-effect studies are presented to examine the specific parameters on the bioeffect and subsequent angiogenic response, both acutely (at 0 day) and at 5 days post exposure. Ultrasound contrast agent concentration is initially explored, followed by a revisit of pressures involvement at a higher contrast agent concentration. In an effort to further understand the biophysical mechanism, a collapse threshold study was conducted, narrowing the pressure range to determine if/ the extent to which collapse was necessary for angiogenesis. Then theoretical models were used to connect the ultrasound contrast agent biophysics to the bioeffects for the therapy. To establish a trail of evidence leading from ultrasound-ultrasound contrast agent induced bioeffects to angiogenesis and characterize the biological motivation, a tissue effect histological study was conducted to establish if the angiogenic response is damage driven. Several other parameters, pulse repetition frequency, exposure duration and the total number of pulses, are also discussed in terms of their influence on the angiogenic response. All of the data gathered herein lead to several conclusions: 1) ultrasound contrast agents increase the angiogenic response, 2) ultrasound contrast agent collapse is required for the increased angiogenic response, 3) increases in vascular permeability occur acutely with collapse, 4) shear stresses during exposure are likely involved in the induction of acute bioeffects, 5) acoustic pressures equal to or greater than 1.3 MPa demonstrate a deleterious effect, and 6) there is a concurrent increase in vascular endothelial growth factor expression and capillary density at 5 days post exposure that suggests an peak range of parameters for ultrasound-ultrasound contrast agent induced angiogenesis. For the studies conducted herein, the optimal window for the angiogenic response was a number of pulses < 3000, a duty factor on the order of 10-2, acoustic pressure ~ 0.7 MPa, pulse repetition frequency ~ 10 Hz, and exposure duration between 2 to 5 minutes. This compilation of studies not only provides some biophysical mechanistic information but also a bit of predictive information when determining appropriate settings for ultrasound-ultrasound contrast agent induced angiogenesis